Glycosylation is one of the most common and structurally diverse forms of protein post-translational modification. Glycans can be N-linked to an asparagine residue, or O-linked to either a serine or a threonine residue. Glycans on any given protein can vary widely in structure, composition and the site of attachment on a protein. Individual sites within one protein type may even contain a heterogeneous population of glycan modifications. This complexity interferes with readily analyzing and characterizing glycosylated proteins. Separating glycans from a glycosylated protein is a common first step in characterizing the glycans present on a glycoprotein. However, this method yields no information about the glycan attachment site on the protein.
Chemical methods exist for capturing glycans, glycopeptides and glycoproteins. For example, hydrazide chemistry (see for example Zhang, et al., Nat. Biotechnol., 21, 660-666 (2003)) involves periodate oxidation of carbohydrate cis-diol groups to dialdehydes and covalent coupling between aldehyde and hydrazide groups to form hydrazine bonds on a solid support. Alternatively, mild periodate treatment can oxidize sialic acids on glycans allowing capture of the oxidized glycoproteins by covalent bonding with hydrazide (see for example, Nilsson, et al., Nature Methods, 6, 809-813 (2009)). However, this method is selective for only glycans with sialic acids and the glycans are also chemically altered by the oxidation process. Ideally, glycomolecule composition is analyzed without chemical modification. Boronic acid reacts with cis-diol-containing saccharides or polyols to form five- or six-membered cyclic esters and this property has been exploited to isolate glycoproteins and glycopeptides. Importantly, the covalent linkage is easily reversible in an acidic pH (Chen, et al., Analyst, 139, 688 (2014)). However, these aforementioned chemical methods unfortunately do not discriminate between N-linked and O-linked glycomolecules.
In titanium dioxide chromatography enrichment, negatively charged sialic acid residues coordinate with the titanium metal ion (for example Larsen, et al., Mol. Cell. Proteomics, 6, 1778-1787 (2007)). However, metal ion affinity chromatography (using titanium, zirconium or silver) is not selective for glycans since negatively charged phosphopeptides or peptides with acidic amino acids, such as glutamic acid and aspartic acid may compete for binding.
HILIC enrichment is a common approach for enriching glycans whereby a water-miscible organic solvent (typically acetonitrile) achieves separation of glycans via a partitioning mechanism. Hydrophilic sugar residues partition into the aqueous phase and are attracted to the hydrophilic groups on a solid support (typically silica or derivatized silica). HILIC materials show broad specificity for glycans but do not discriminate between O-glycan linked and N-glycan linked glycomolecules (Chen, et al., Analyst, 139, 688 (2014)).
Finally, lectins are proteins with natural carbohydrate binding properties. Many lectins have been characterized and several have been employed for glycopeptide/glycoprotein enrichment or detection. Lectins recognize the variable region of N-linked glycans and the specificity of a lectin may be quite narrow (L-phytohemagglutinin (L-PHA) for the targeted beta-1,6-branched N-linked glycan (see for example, Ahn, et al., Anal. Chem., 82, 4441-4447 (2010)) or relatively broad in the case of Concanavalin A, which recognizes a high mannose structure. Nevertheless, a diverse set of lectins with selective affinities for specific carbohydrate epitopes has been used to investigate the human glycoproteome. However, to this date no single lectin has been shown to possess sufficient selectivity to analyze the entire N-glycan linked glycoproteome. Another major drawback of existing lectin based enrichment methods is low affinity of most natural lectins for their substrates (Kd ranging from 10 mM to 1 μM, (see for example, Fanayan, et al., Electrophoresis, 33, 1746-1754 (2012)). Elution of bound glycomolecules from lectins may be achieved by low pH, for example glycine-HCl buffer (at pH 2-2.8) or 100 mM acetic acid; yet low pH exposure can potentially alter glycan structure. Alternatively, glycomolecule elution from lectins can be accomplished using the appropriate sugar to displace the bound glycomolecule from the immobilized lectin but the added sugar will complicate most downstream analyses.
Typically mass spectrometry analysis of a sample having a mixture of peptides and glycopeptides (both O-linked and N-linked) reveals a highly complex pattern of peaks. The primary problem is that this complex pattern cannot be interpreted to identify and characterize the individual glycomolecules in the sample. Therefore, a need exists for an enrichment reagent that is able to selectively isolate an individual class of glycomolecule, for example either N-glycan linked glycomolecules or O-glycan linked glycomolecules. Upon fractionation of a complex sample, the results of the mass spectrometry analysis might be more easily interpreted.
Many important biological activities are affected by protein glycosylation, including protein folding, protein metabolism, protein-protein interactions, immune cell recognition and intercellular signaling. Given the emerging interest in glycoproteins as biomarkers, a need exists for readily analyzing and characterizing protein glycosylation.